JP5430803B2 - Peptides and angiotensin converting enzyme inhibitors - Google Patents

Description

  The present invention relates to a novel peptide and an angiotensin converting enzyme inhibitor containing the peptide. An angiotensin converting enzyme inhibitor can be used as food, feed, and medicine.

  Angiotensin converting enzyme (ACE) is an enzyme that acts on angiotensin I generated from angiotensinogen by cleavage with renin, liberates two C-terminal amino acids, and converts them into angiotensin II. Angiotensin converting enzyme generates angiotensin II having a strong pressor action and also has an action of inactivating bradykinin having a hypotensive action. Because of these actions, angiotensin converting enzyme inhibitors are used as therapeutic agents for hypertension, and captopril, renibase, lisinopril and the like are known as commercially available drugs. In addition, angiotensin converting enzyme inhibitors are recommended to be actively used as first-line drugs for many pathologies such as heart disease, kidney disease, cerebrovascular disorder, arteriosclerotic disease, diabetes, metabolic syndrome, and elderly hypertension. (High Blood Pressure Treatment Guidelines 2009).

  On the other hand, peptides having an angiotensin converting enzyme inhibitory action have been found in natural products. Since these are composed of natural amino acids, they can be taken orally as nutritional sources, as well as exhibiting blood pressure lowering effects. It is used as a health food. For example, peptides (Patent Documents 1 to 11 and Non-Patent Document 1) obtained by degrading casein with an enzyme are examples. In addition, many other natural peptides having an angiotensin converting enzyme inhibitory action are known (Non-patent Document 2).

JP 58-109425 A JP 59-44323 JP 59-44324 A JP 61-36226 A JP 61-36227 A JP-A-6-277090 JP-A-6-277091 JP-A-6-279491 Japanese Patent Laid-Open No. 7-101982 JP 7-101985 A Japanese Patent No. 3816921

Maruyama, S. et al., Agric. Biol. Chem. 49 (5), 1405-1409, 1985 Fang, H. et al., PEPTIDES, 29, 1062-1071, 2008

As described above, various peptides having an angiotensin converting enzyme inhibitory action are known, but these peptides still have insufficient angiotensin converting enzyme inhibitory activity as a function in food. Therefore, it is desired to obtain a natural product-type peptide having higher angiotensin converting enzyme inhibitory activity and to apply it to food or medicine.
An object of the present invention is to provide a novel peptide having an angiotensin converting enzyme inhibitory action and an angiotensin converting enzyme inhibitor containing the same.

  The present inventors have intensively studied to solve the above problems. That is, by synthesizing a hexapeptide in which lysine is fixed on the C-terminal side and 5 types of amino acids (arginine, leucine, isoleucine, proline, serine) are randomly bound to the peptide, high angiotensin converting enzyme inhibition is observed in the mixture. It has been found that there is a novel peptide having activity, and that the peptide has a specific sequence, and the present invention has been completed.

The present invention provides a peptide consisting of the following amino acid sequence.
Xaa-Arg-Xaa-Pro-Ser-Lys (SEQ ID NO: 1)
(However, each amino acid is L-form, and Xaa is independently Ile or Leu.)
The peptide of the present invention preferably has any of the following amino acid sequences.
a) Ile-Arg-Ile-Pro-Ser-Lys (SEQ ID NO: 2)
b) Ile-Arg-Leu-Pro-Ser-Lys (SEQ ID NO: 3)
c) Leu-Arg-Leu-Pro-Ser-Lys (SEQ ID NO: 4)
d) Leu-Arg-Ile-Pro-Ser-Lys (SEQ ID NO: 5)
The present invention also provides an angiotensin converting enzyme inhibitor containing the peptide as an active ingredient.
The present invention also provides a blood pressure lowering agent containing the peptide as an active ingredient.
The present invention also provides a method for lowering blood pressure in a subject comprising administering the peptide to a subject in need of blood pressure reduction.
The present invention also provides use of the peptide for lowering blood pressure in a subject in need of blood pressure reduction.

The separation chromatogram by the reverse phase HPLC of a synthetic peptide mixture. The horizontal axis represents time (minutes), and the vertical axis represents absorbance at 215 nm.

Next, a preferred embodiment of the present invention will be described in detail. However, the present invention is not limited to the following preferred embodiments, and can be freely changed within the scope of the present invention. In the present specification, percentages are expressed by mass unless otherwise specified.
The peptide of the present invention has a sequence represented by Xaa-Arg-Xaa-Pro-Ser-Lys (SEQ ID NO: 1). The peptide of the present invention may be a salt of this peptide. In the present invention, Leu is an L-leucine residue, Arg is an L-arginine residue, Ile is an L-isoleucine residue, Pro is an L-proline residue, Ser is an L-serine residue, and Lys is an L-lysine residue. Indicates a group. Xaa is independently Ile or Leu.

That is, the peptide of the present invention has any of the following amino acid sequences.
a) Ile-Arg-Ile-Pro-Ser-Lys (SEQ ID NO: 2)
b) Ile-Arg-Leu-Pro-Ser-Lys (SEQ ID NO: 3)
c) Leu-Arg-Leu-Pro-Ser-Lys (SEQ ID NO: 4)
d) Leu-Arg-Ile-Pro-Ser-Lys (SEQ ID NO: 5)

  The peptide of the present invention can be produced by chemical synthesis. The chemical synthesis of the peptide of the present invention can be carried out by a liquid phase method or a solid phase method usually used for the synthesis of oligopeptides. The synthesized peptide is deprotected as necessary to remove unreacted reagents, by-products and the like. Such peptide synthesis can be performed using a commercially available peptide synthesizer. The peptide of the present invention is preferably isolated and purified from the above synthesized (mixed) product. Peptide purification is usually carried out in the same manner as that used for oligopeptide purification, such as ion exchange chromatography, adsorption chromatography, reverse phase chromatography, partition chromatography, gel filtration chromatography and other various chromatography methods. , Solvent precipitation, salting out, and partitioning between the two liquid phases, and the like. In the purification of the peptide of the present invention, the fraction containing the target substance can be determined using the angiotensin converting enzyme inhibitory action described below as an index, and the active component of these fractions can be determined by mass spectrometry or / and a protein sequencer. Can be identified.

  The peptide of the present invention can also be produced by expressing a recombinant DNA encoding the peptide in an appropriate host cell. As vectors and hosts necessary for the production of recombinant DNA, those usually used for the production of proteins and peptides can be used.

The peptide of the present invention can be used as an active ingredient of an angiotensin converting enzyme inhibitor. The peptide of the present invention has an angiotensin converting enzyme inhibitory action. When an angiotensin converting enzyme is inhibited, the production of angiotensin II and the inactivation of bradykinin by the enzyme are suppressed, resulting in a blood pressure lowering effect. Therefore, the peptide or angiotensin converting enzyme inhibitor of the present invention is a preventive or therapeutic agent for various diseases derived from hypertension such as cerebral hemorrhage, cerebral infarction, angina pectoris, myocardial infarction, renal failure, etc., specifically blood pressure. It can be used as a depressant. That is, one form of the present invention is a blood pressure lowering agent containing the peptide of the present invention as an active ingredient. Moreover, an angiotensin converting enzyme inhibitor is known to have an effect on essential hypertension of unknown cause, and the peptide of the present invention is expected to show a therapeutic or preventive effect on essential hypertension. In addition, it can also be used as a therapeutic or prophylactic agent for diseases such as cardiac hypertrophy and angina which are known to be effective for angiotensin converting enzyme inhibitors.
Another aspect of the present invention is a method for lowering blood pressure in a subject comprising administering a peptide of the present invention to a subject in need of blood pressure reduction.
Another aspect of the present invention is the use of a peptide of the present invention for lowering blood pressure in a subject in need of blood pressure reduction.
The subject requiring blood pressure lowering is not particularly limited as long as it is effective to inhibit angiotensin converting enzyme, but patients having various diseases derived from the above-mentioned hypertension, patients with essential hypertension, and cardiac hypertrophy And patients having diseases such as angina. Also, in these methods and uses, “decrease in blood pressure” can be “treatment of disease”.

The peptide of this invention can be mix | blended with a pharmaceutical, food-drinks, feed, cosmetics, a quasi-drug, etc. One type of peptide may be blended, or a mixture of any two or more types may be used.
For example, additives such as carriers, excipients, binders, disintegrants, lubricants, colorant stabilizers, flavoring agents, diluents, and solvents for injections can be used for pharmaceutical formulation. Specific preparations include tablets (including sugar-coated tablets, enteric-coated tablets, buccal tablets), powders, capsules (including enteric capsules and soft capsules), granules (including those coated), pills, Illustrative examples include troches, encapsulated liposomes, solutions, and pharmaceutically acceptable sustained release formulations.

Carriers and excipients used in these formulations include lactose, glucose, sucrose, mannitol, potato starch, corn starch, calcium carbonate, calcium phosphate, calcium sulfate, crystalline cellulose, licorice powder, gentian powder and the like as binders Examples thereof include starch, gelatin, syrup, polyvinyl alcohol, polyvinyl ether, polyvinyl pyrrolidone, hydroxypropyl cellulose, ethyl cellulose, methyl cellulose, carboxymethyl cellulose and the like.
Examples of the disintegrant include starch, agar, gelatin powder, sodium carboxymethyl cellulose, carboxymethyl cellulose calcium, crystalline cellulose, calcium carbonate, sodium bicarbonate, and sodium alginate.
Furthermore, as the lubricant, magnesium stearate, hydrogenated vegetable oil, macrogol, etc., and as the colorant, red No. 2, yellow No. 4, and blue No. 1, etc., which are allowed to be added to pharmaceuticals, etc. , Respectively.
In addition, as for stabilizers, flavoring agents, diluents, solvents for injections, and the like, components usually used in the production of pharmaceuticals can be used.

  Tablets and granules are sucrose, hydroxypropylcellulose, purified shellac, gelatin, sorbitol, glycerin, ethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, cellulose phthalate acetate, hydroxypropylmethylcellulose phthalate, methyl methacrylate as necessary And a methacrylic acid polymer.

  The angiotensin converting enzyme inhibitor or blood pressure lowering agent of the present invention may be administered either orally or parenterally, but oral administration is preferred. Parenteral administration includes intravenous injection, rectal administration, inhalation and the like. Examples of the dosage form for oral administration include tablets, capsules, troches, syrups, granules, powders, ointments and the like. Moreover, the medicine and pharmaceutical composition which have the angiotensin converting enzyme inhibitory action known or discovered in the future can also be used together with the peptide of this invention. The drug or pharmaceutical composition to be used in combination may be contained as one of the active ingredients in the drug of the present invention, or it is not contained in the drug of the present invention and is combined with the drug of the present invention as a separate drug. May be commercialized.

Further, the peptide of the present invention can be contained in food as an active ingredient, and processed as a food having an angiotensin converting enzyme inhibitory action or blood pressure lowering action as one embodiment of an angiotensin converting enzyme inhibitor or blood pressure lowering agent. .
Such foods may be in the form of liquids, pastes, solids, powders, etc., in addition to tablet confections, liquid foods, feeds (including for pets), etc., for example, bread, macaroni, spaghetti, noodles, cakes Flour products such as mix, fried flour, bread crumbs, instant noodles, cup noodles, retort / cooked food, cooked canned food, microwave food, instant soup / stew, instant miso soup / soup, canned soup, freeze-dried food, other instant foods Instant foods such as foods; Canned agricultural products, canned fruits, jams and marmalades, pickles, boiled beans, dried agricultural products, processed cereals (cereal processed products); canned marine products, fish ham and sausages, fish paste products, Processed marine products such as marine delicacy, tsukudani, etc .; Livestock canned and pasted products, livestock processed products such as livestock ham and sausage; processed milk, milk drinks, yogurts Milk and dairy products such as lactic acid bacteria beverages, cheese, ice cream, formula milk powder, cream and other dairy products; fats and oils such as butter, margarine, vegetable oil; soy sauce, miso, sauces, tomato processed seasonings , Mirins, basic seasonings such as vinegar; cooking mixes, curry ingredients, sauces, dressings, noodle soups, spices, and other complex seasonings and foods; frozen foods, Semi-cooked frozen foods, frozen foods such as cooked frozen foods; caramel, candy, chewing gum, chocolate, cookies, biscuits, cakes, pie, snacks, crackers, Japanese confectionery, rice confectionery, bean confectionery, dessert confectionery, other confectionery Sweets: carbonated drink, natural fruit juice, fruit juice drink, soft drink with fruit juice, fruit drink, fruit drink with fruit granules, vegetable drink, soy milk, soy milk drink Coffee beverages, tea beverages, powdered beverages, concentrated beverages, sports beverages, nutritional beverages, alcoholic beverages, other beverages such as other favorite beverages, baby food, sprinkles, other marketed foods such as tea paste, etc. Examples include formula milk powder; enteral nutrition; functional food (food for specified health use, functional food for nutrition).

Furthermore, as an embodiment of an angiotensin converting enzyme inhibitor or blood pressure lowering agent, the peptide of the present invention can be contained in the feed as an active ingredient, and processed as a feed having an angiotensin converting enzyme inhibitory action or blood pressure lowering action. .
The form of the feed is not particularly limited. For example, the peptide powder of the present invention or an aqueous solution thereof (syrup or the like) is used for cereals such as corn, wheat, barley, rye, and milo; soybean oil cake, rapeseed oil cake, coconut oil cake Vegetable oils such as flaxseed, flaxseed oil, rice bran, rice bran, defatted rice bran, etc .; production corn such as corn gluten meal, corn jam meal; fish meal, skim milk powder, whey, yellow grease, tallow, etc. Animal feeds; yeasts such as torula yeast and beer yeast; mineral feeds such as tricalcium phosphate and calcium carbonate; fats and oils; simple amino acids; sugars and the like. Examples of the form of the feed include pet food, livestock feed, and fish feed.

  In the angiotensin converting enzyme inhibitor or antihypertensive agent of the present invention (each aspect of pharmaceuticals, foods and drinks, feeds, cosmetics, quasi drugs, etc.), the amount of the peptide of the present invention (the total amount in the case of multiple peptides) ) Is preferably 0.001% by mass or more, more preferably 0.01% by mass or more, and 0.1% by mass with respect to the final composition of the angiotensin converting enzyme inhibitor or blood pressure lowering agent. The above is particularly preferable.

  The dose of the angiotensin converting enzyme inhibitor or blood pressure lowering agent of the present invention varies depending on age, symptoms, etc., but is usually 0.001 to 3000 mg / day, preferably 0.01 to 30 mg / day as the amount of the peptide of the present invention. It may be administered once a day or divided into 2 to 3 times a day. In addition, when the angiotensin converting enzyme inhibitor or blood pressure lowering agent of the present invention is ingested or taken, the effects of the present invention are sufficiently exhibited at any timing before, between, and after a meal.

  Hereinafter, the present invention will be described in more detail using examples, but the present invention is not limited to these examples.

<Example 1>
(1) Chemical synthesis of peptides Using a peptide synthesizer (Model 433A type, Applied Biosystems), a mixture of five amino acid derivatives [Fmoc-L-Leu, Fmoc-L-Arg (Mtr), Fmoc-L-Ile , Fmoc-L-Ser (tBu), Fmoc-L-Pro], Fmoc-L-Lys (Boc) -Wang Resin (both domestic chemistry) as raw materials, AA (6)- AA (5) -AA (4) -AA (3) -AA (2) -Lys [AA (n) is one of five amino acids, and 5 ⁵ = 3125 peptides are synthesized] ( A mixture of SEQ ID NO: 6) was synthesized. The operation was performed according to the manual of Applied Biosystems. After the synthesis reaction, it was deprotected. The obtained peptide was purified under the following HPLC conditions.

(2) Separation of peptides by HPLC The synthesis mixture was separated by reverse phase HPLC. The HPLC conditions are shown in the following HPLC condition 1.

[HPLC condition 1]
Column: Cadenza CD-18 10 mmI. D. × 250mm (manufactured by Intact)
Detection: UV 215nm
Flow rate: 3 ml / min Eluent A: Aqueous solution containing 0.1% TFA Eluent B: Acetonitrile solution containing 0.1% TFA

After the sample is introduced into the column, the eluent (A / B) is maintained at 98/2 for 5 minutes, to the eluent (75/25) for 30 minutes, to the eluent (50/50) for 10 minutes, and further to the eluent ( Gradient elution was performed in 3 minutes until 20/80), and then the synthetic peptide mixture was separated under a gradient condition of maintaining the eluent (20/80) for 5 minutes (chromatogram: FIG. 1).
The eluate was fractionated for each peak, and the angiotensin converting enzyme inhibitory ability was measured by the method described later, and the fractions having strong angiotensin converting enzyme inhibitory ability were retention times of 34.0 minutes, 34.4 minutes, 34.7 minutes, and 35.1. Eluted in minutes.

  The peptides contained in these fractions were analyzed with a protein sequencer (PPSQ-23A) manufactured by Shimadzu Corporation. As a result, Ile-Arg-Ile-Pro-Ser-Lys (hereinafter referred to as HP1), Ile-Arg-Leu-Pro-Ser-Lys (hereinafter referred to as HP2), Leu-Arg-Leu-Pro-Ser-Lys (hereinafter referred to as HP3), and Leu-Arg-Ile-Pro- Ser-Lys (hereinafter referred to as HP4).

<Example 2>
[Angiotensin-converting enzyme inhibitory action of peptides]
(1) Test method Angiotensin converting enzyme inhibition was measured according to the method of Kashman et al. [Biochemical pharmacology, Vol. 20, pages 1637 to 1648 (1971)].
As a sample, the peptide of the present invention (HP1, HP2, HP3, HP4) obtained in Example 1, the peptide described in Agricultural and Biological Chemistry 49 (5), 1405-1409, 1985 (Phe-Phe-Val-Ala- Pro-Phe-Pro-Glu-Val-Phe-Gly-Lys: SEQ ID NO: 7) and a peptide (Met-Lys-Pro) described in Japanese Patent No. 38169921 (WO2003 / 044044)) were used. All of these peptides were chemically synthesized in the same manner as in Example 1.

  The sample was dissolved in 0.1 M borate buffer (containing 0.3 M NaCl, pH 8.3), 0.08 ml was placed in a test tube, and then 0.1 M borate buffer (containing 0.3 M NaCl). 0.2 ml of an enzyme substrate (Hiprilhistidylleucine, Sigma) adjusted to 5 mM at pH 8.3) was added, and the mixture was incubated at 37 ° C. for 3 minutes. Subsequently, 0.02 ml of rabbit lung angiotensin converting enzyme (manufactured by Sigma) prepared to be 0.1 U / ml by adding distilled water was added and reacted at 37 ° C. for 30 minutes.

  Thereafter, 0.25 ml of 1N hydrochloric acid was added to complete the reaction, 1.7 ml of ethyl acetate was added, and the mixture was vigorously stirred for 20 seconds, centrifuged at 3000 rpm for 10 minutes, and 1.4 ml of the ethyl acetate layer was collected. did. The obtained ethyl acetate layer was heated to remove the solvent, and then 1.0 ml of distilled water was added, and the absorption of the extracted hyprilic acid (absorbance at 228 nm) was measured to determine the enzyme activity.

  The inhibitory activity was calculated from the following formula, and IC50 [sample concentration (μM) necessary for inhibiting 50% of the activity of angiotensin converting enzyme] was determined.

Inhibition rate = (A−B) / (A−C) × 100%
A: Enzyme activity when sample (peptide) is not included (absorbance at 228 nm)
B: Enzyme activity when adding sample (absorbance at 228 nm)
C: Enzyme activity when no enzyme and sample are added (absorbance at 228 nm)

(2) Test results The test results are shown in Table 1. As is clear from Table 1, all of the peptides of the present invention (HP1, HP2, HP3, HP4) were found to have potent angiotensin converting enzyme inhibitory activity.

<Example 3>
[Antihypertensive action of peptides in animals]
(1) Test method 14 12-week-old SHR / Hos male rats (purchased from Japan SLC Co., Ltd.) were preliminarily raised for 1 week, and the blood pressure of the rats was measured using a small animal non-invasive automatic sphygmomanometer (MK-2000, Muromachi Kikai Co., Ltd.).

  Using the systolic blood pressure as an index, the group was divided into two groups so that the average systolic blood pressure before administration for each group was almost the same value, and then the animals were fasted for about 14 hours after being divided into two groups. In Example 1, the peptide obtained in Example 1 (Leu-Arg-Ile-Pro-Ser-Lys: HP4) was dissolved in water for injection and orally administered to rats at a rate of 5 mL / kg body weight (3 mg / kg body weight as HP4). The blood pressure of the rats was measured immediately before sample administration, 1, 3, 6 and 8 hours after sample administration.

In the control group, the same volume of water for injection was orally administered instead of the HP4 aqueous solution, and the blood pressure of the rats was measured immediately before sample administration, 1, 3, 6 and 8 hours after sample administration.
(2) Test results

The test results are shown in Table 2. As is clear from Table 2, blood pressure was continuously decreased in the test group (HP4 administration group), but not in the control group. Therefore, it was found that the peptide of the present invention (Leu-Arg-Ile-Pro-Ser-Lys: HP4) has a blood pressure lowering effect on animals.
In addition, as a result of conducting the same test also about the other peptides (HP1, HP2, HP3) obtained in Example 1, it was found to have a blood pressure lowering effect like HP4.

  According to the present invention, a novel peptide having an angiotensin converting enzyme inhibitory action is provided. An angiotensin converting enzyme inhibitor containing the peptide can be used as a medicine. Since the peptides of the present invention are all composed of natural (L) amino acids, they are highly safe and can be used in foods.

Claims (9)

A peptide consisting of the following amino acid sequence.
Xaa-Arg-Xaa-Pro-Ser-Lys
(However, each amino acid is L-form, and Xaa is independently Ile or Leu.)   The peptide of Claim 1 which is a peptide which consists of Ile-Arg-Ile-Pro-Ser-Lys.   The peptide of Claim 1 which is a peptide which consists of Ile-Arg-Leu-Pro-Ser-Lys.   The peptide of Claim 1 which is a peptide which consists of Leu-Arg-Leu-Pro-Ser-Lys.   The peptide of Claim 1 which is a peptide which consists of Leu-Arg-Ile-Pro-Ser-Lys.   An angiotensin converting enzyme inhibitor containing the peptide according to any one of claims 1 to 5 as an active ingredient.   The antihypertensive agent which contains the peptide as described in any one of Claims 1-5 as an active ingredient. A method for lowering blood pressure of a subject, comprising administering the peptide of any one of claims 1 to 5 to a subject (except a human) in need of blood pressure lowering. Use of the peptide according to any one of claims 1 to 5 for lowering blood pressure of a subject (excluding humans) in need of blood pressure reduction.

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